The long-term objective of this project is to provide a complete and detailed understanding of the beta-oxidation of unsaturated and polyunsaturated fatty acids. This knowledge will be important for analyzing and interpreting abnormalities of fatty acid metabolism caused by enzyme deficiencies, ischemia, diabetes, and dietary factors. The focus of this study is the reactions and auxiliary enzymes that are unique to the beta-oxidation of unsaturated fatty acids. The contributions of the isomerase-dependent and reductase-dependent pathways to the beta-oxidation of unsaturated fatty acids with odd-numbered double bonds will be investigated with rat mitochondrial extracts and intact mitochondria. For the latter experiments, inhibitors will be used or developed that inactivate specific enzyme, e.g. delta3,5,delta2,4-dienoyl-CoA isomerase, in intact mitochondria. Delta3,delta2-Enoyl-CoA isomerases, which control the flux through the reductase-dependent pathway, will be studied to answer questions about the existence of isozymes and their substrate specificities. The dual function of delta3,5,delta2,4-dienoyl-CoA isomerase as a dienoyl-CoA isomerase and a trienoyl-CoA isomerase will be investigated by site-specific mutagenesis of the enzyme. Efforts to solve the crystal structure of 2,4-dienoyl-CoA reductase from E.coli are underway and will continue. Finally, the intramitochondrial accumulation of beta-oxidation intermediates of trans fatty acids with odd-numbered double bonds will be analyzed and the possible harmful effects of these intermediates on cell viability will be investigated.